Method for manipulating magnetic particles in a liquid medium

ABSTRACT

A method of mixing magnetic particles ( 3 ) in a reaction chamber ( 2 ) that is part of a microfluidic device and that contains the said particles in suspension, comprises the steps: (a) providing an electromagnetic means ( 1,1′,6,7 ) to generate magnetic field sequences having polarity and intensity that vary in time and a magnetic field gradient that covers the whole space of the said reaction chamber ( 2 ); (b) applying a first magnetic field sequence to separate or confine the particles ( 3 ) so the particles occupy a sub-volume in the volume of the reaction chamber ( 2 ); (c) injecting a defined volume of the said reagent in the reaction chamber; and (d) applying a second magnetic field sequence to leads the particles ( 3 ) to be homogenously distributed and dynamically moving over a substantial portion of the whole reaction chamber volume.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/537,153, filed on Nov. 10, 2014; which is a continuation of U.S.patent application Ser. No. 12/340,018, filed on Dec. 19, 2008, now U.S.Pat. No. 8,999,732, issued on Apr. 7, 2015; which is acontinuation-in-part of International Application No. PCT/IB2007/052409filed on Jun. 21, 2007, which claims priority under 35 U.S.C. § 365(b)to International Application No. PCT/IB2006/052005, filed on Jun. 21,2006 and which claims priority under 35 U.S.C. § 365(b) to InternationalApplication No. PCT/IB2006/054182 filed on Nov. 9, 2006; and which isalso a continuation-in-part of International Application No.PCT/IB2007/052410 filed on Jun. 21, 2007, which claims priority under 35U.S.C. 365(b) to International Application No. PCT/IB2006/052005, filedon Jun. 21, 2006 and which claims priority under 35 U.S.C. § 365(b) toInternational Application No. PCT/IB2006/054182 filed on Nov. 9, 2006.The contents of each of these applications are herein incorporated byreference in their entireties.

FIELD OF THE INVENTION

The invention relates to a method of handling and mixing magneticparticles within a reaction chamber that is a part of a fluidic ormicrofluidic platform. More particularly, the invention concerns amethod of handling magnetic particles in a way to improve the mixing ofthe particles with the surrounding liquids medium and where the liquidsare automatically handled in a fluidic platform. Further, the inventionrelates to a method for conducting assays on a test sample containingspecific biological or chemical substances using active bio-chemicallysurface magnetic particles and where the particles are handled followingthe foregoing system and method.

DESCRIPTION OF RELATED ARTS

Nowadays, magnetic particle (bead) is a standard technology inbiochemical assays and diagnostics. Magnetic particle technology isindeed a robust technology that allows achieving high performances(sensitivity and accuracy) and also opens the possibility of easyautomation of assay protocols. For many applications, the surface ofmagnetic particles is coated with a suitable ligand or receptor, such asantibodies, lectins, oligonucleotides, or other bioreactive molecules,which can selectively bind a target substance in a mixture with othersubstances. Examples of small magnetic particles or beads are disclosedin U.S. Pat. Nos. 4,230,685, 4,554,088 and 4,628,037.

One key element in magnetic particles bio-separation and handlingtechnology is an efficient mixing to enhance the reaction rate betweenthe target substances and the particle surfaces. Indeed, as for anysurface-based assay the reaction is strongly limited by the naturaldiffusion process, a strong steering and mixing is necessary to promotethe affinity binding reaction between the ligand and the targetsubstance.

A typical example of magnetic particles mixing apparatus in test mediumis disclosed in U.S. Pat. No. 6,231,760 and commercially available bySigris Research Inc. under the name of MIXSEP™ system. In this patentand system, the test medium with the magnetic particles in a suitablecontainer are placed in a magnetic field gradient generated by anexternal magnet. The mixing concept is based on either the movement of amagnet relative to a stationary container or movement of the containerrelative to a stationary magnet using mechanical means, thereforeinducing a “relative displacement” of the magnetic gradient positionwithin the container. This magnetic field gradient displacement will inturn induce the magnetic particles to move continuously with the changeof the magnet (magnetic field gradient) position, thereby effectingmixing. However, with this method the magnetic field gradient willattract and confine the particles in a cavity region close to the wallsof the container. In such condition, the contact between the particlesand the test medium is limited to the said cavity space which reducesthe mixing efficiency. Although the “mechanical movement” of magnets isclaimed as a mixing means, also described is the possibility ofproducing angular movement of the particles by sequential actuation ofelectromagnets disposed around the container. However, whileelectromagnets provide a much lower magnetic field when compared withpermanent magnets, as described the magnetic coupling between adjacentelectromagnets strongly repel the magnetic flux outside the containerresulting in a further reduction of the magnetic field intensity andintensification of the cavity effect. Under such condition, theparticles agitation (movement) and mixing will be strongly alteredleading the particles to slowly move, mostly, as aggregates at theregion close the walls border.

Within the same spirit, in the U.S. Pat. No. 6,764,859 a method ofmixing magnetic particles in a container is disclosed based on relative“mechanical” movement between the container and intervening arraygeometry of magnet. In such configuration the adjacent magnets haveopposite polarity which induces a change of magnetic field polarityduring the relative intervening movement between the container and twoadjacent magnets. In such conditions indeed, the particles can be movedwhile relatively separated from each other, which will potentiallyaffect the mixing. However, in this approach when one takes inconsideration the whole duration of the particles handling process, thetime during which the particles are relatively separated from each otheris relatively short. As a consequence, several mixing cycles arenecessary to assure effective mixing. Moreover, during the mixingprocess the particles are not homogenously contacted with the samplevolume in the test tube, which will in turn strongly limit the mixingefficiency. This issue is more pronounced as the sample volume is large.

Consequently when these mechanical mixing approaches are compared withthe “manual shaking of a test tube”, the reaction time and theperformance are substantially similar if not lower, indicating thatdiffusion is still an important limiting factor.

Other aspects for magnetic particles separation and resuspending aredisclosed in E.P. Pat 0,504,192. This patent discloses the use ofsequential actuation of two magnetic field sources (electromagnets)disposed opposite to each other at the walls of a chamber. The proposedactuation concept of the said electromagnets is based on sequentialenergizing (actuation) of the electromagnets by “binary” (i.e., on andoff) or “analog” in which a first electromagnet is gradually fullyenergized, and then has its power reduced, while the next electromagnetis gradually energized, and so on. Through this actuation the particleswill be moved and drawn to the reaction chamber volume and therebyresuspended. While the concept of using (at least) two electromagnetswith “sequential” actuation is conceptually an evident manner forparticles resuspension from an aggregate, during their “movement” theparticles remain mostly agglomerated due to their dipolar interactionunder the applied magnetic field. The only way, after moving the“super-paramagnetic” particles to occupy the chamber volume, to fullyassure “homogenous” resuspension in the chamber is to completely removethe external magnetic field and leave the desegregation to Brownian andthermal agitations. Additionally, the application discloses thatalternately energizing and de-energizing the two electromagnets at asufficiently rapid rate keeps the particles suspended in the center ofthe chamber. This process limits the movement of the particles to arelatively small distance, significantly reducing the mixing efficiencybetween particles and the surrounding liquid medium.

In general, beyond the limited mixing capability of the state of the artmagnetic particles technologies, mainly based on the concept of“bringing a magnet in the proximity of a test tube”, the integration andthe automation of magnetic particles assay procedures are very complex,necessitating bulky robotic systems. These limitations become all themore critical as the assay procedures are becoming more and morecomplex.

Microfluidics based technology is nowadays perceived as an emergingtechnology with a great potential that can lead to easier integration ofcomplex bio-chemical assay procedures in an easy-to-use and miniaturizedautomated system. Combining magnetic particles technology withmicrofluidics will certainly be of great importance as the precisecontrol of different reagents (allowed by microfluidics) and handling ofbiological species and their reactions (allowed by magnetic particles)will be integrated together within a single system.

One approach of mixing magnetic particles in a microfluidics channel istaught in the publication “Magnetic Force Driven Chaotic Micro-Mixer”,by Suzuki, H, in the proceedings of The Fifteenth IEEE InternationalConference on Micro Electro Mechanical Systems, 2002. The approachconsists in flow mixing of magnetic particles injected in suspension ina microfluidic channel and where the mixing with the surrounding mediumis assured by a magnetic field generated by embeddedmicro-electromagnets along the flow path. The combination of themagnetic force induced by the micro-electromagnets on the particlesalong with the flow driving force in the microchannel induces a chaoticregime and thereby mixing. A similar concept has been recently disclosedin U.S. patent application number 2006/140,051 where the magnetic fieldis generated by electromagnets disposed on the sidewalls in apredetermined direction with respect to the direction of the flow. Byturning off/on the electromagnets in sequential operation, a rotatingmagnetic force can be created leading to mixing of the particles carriedby the flow. The major limitation of this “in flow mixing” approach isthat the volume of the test medium that can be mixed with the particlesis very small and the reaction time very short, limiting considerablythe cases of its applicability.

To overcome the limitations of the “in-flow mixing approach”, a solutionconsists in retaining the particles in a given location of a fluidicchannel or chamber using a magnetic field gradient while the test mediumis injected with a flow through the retained magnetic particles. Thisapproach has been disclosed in the U.S. patent applications number2005/032,051 and 2004/166,547 where the particles retained in a flowmicrochannel have been used as a solid support for immunoassayprocedures. Along the same lines, a flow-through concept applied for DNAhybridization and detection assay is described in the publication:“Dynamic DNA hybridization on a Chip Using Paramagnetic Beads”, by Z.Hugh Fan & al., Analytical Chemistry, 71, 1999. However the so describedflow-through approach suffers from a serious physical constraint, sincein order to be handled in an environment with continuous fluidicprocessing, the particles must be continuously exposed to a magneticfield. Under such conditions the particles will stick together andagglomerate thereby losing their main advantage: the particle surfacethat is in active contact with the fluid flow will be drasticallyreduced which will seriously compromise the assay performance.

A solution to the agglomeration problem of magnetic particles in theflow-through approach has been disclosed U.S. patent application2005/208,464. In this approach, the particles are retained in a portionof a flow-channel to form a kind of a filter that substantiallyhomogenously covers the flow-channel cross section. To obtain thisfilter, the magnetic particles are manipulated using a time-varyingfield (amplitude, frequency and polarity) to control the particlesagglomeration. The efficiency of this approach for microfluidic mixingof liquids has been demonstrated in a publication from the same authorgroup “Manipulation of Self-Assembled Structures of Magnetic Beads forMicrofluidic Mixing and Assaying”, by A. Rida & al. AnalyticalChemistry, 76, 2004. Even demonstrating an important development inmagnetic particles handling and mixing in a microfluidic environment,the approach disclosed in U.S. patent application 2005/208,464 suffershowever from many practical limiting constraints. First, as theparticles are kept stationary and fixed in a narrow segment of theflow-through cell, the contact between the particles and the targetsubstance is limited to that narrow region and for a very short time,which in practice makes such process difficult to set up. Secondly, thisapproach is specifically adapted for handling and mixing magneticparticles under flow-through conditions in microfluidics environments,which make it not fully adapted for different assay conditions.

The applicable known procedures and approaches have shortcomings,including the requirement for handling and mixing magnetic particles invarious environments with more focus on microfluidics, as well variousprocess constraints, limiting factors and inefficiencies.

SUMMARY OF THE INVENTION

The present invention provides devices and methods for handling andefficiently mixing magnetic particles in fluidic environments. “Mixing”in the present context means in particular contacting in a veryefficient manner large particles surfaces with the surrounding liquidmedium, in such a way as to achieve: (1) an effective binding of theparticles to a certain target molecule(s) and (2) further possibilitiesto wash, separate, elute and detect the targets captured on theparticles from the residual liquid medium.

The proposed mixing mechanism provides a considerable and perpetualincrease of the active surface of particles par unit of volume leadingto an enhanced contact between this large surface of particles and thetarget substances. Further, the proposed magnetic particles handlingprocess advantageously assures a homogenous mixing coveringsubstantially the whole reaction volume in a fraction of time allowingthereby much more sample volume to be effectively and rapidly contactedwith the particle surfaces, Moreover, during their manipulation theparticles are in perpetual effective movement covering the wholereaction chamber volume, which is a key in enhancing particles mixing.

Further, the invention provides new devices and methods that practicallyallow the integration of complex assay procedures in a compact and easyto use system that can operate under flow-through or, advantageously,under non flow-through conditions.

Accordingly, a main aspect of the invention concerns a method formanipulating and mixing magnetic particle in microfluidics environments,attained according to independent claim 1.

Accordingly, a main aspect of the invention concerns a method formanipulating and mixing magnetic panicle in microfluidics to extracttarget molecule(s) that enter in the composition of intracellularcomplexes in a sample volume, attained according to independent claim12.

Different embodiments are set out in the dependent claims.

According to one embodiment of the present invention, a method forreagents processing and mixing magnetic particles with the said reagentsin a reaction chamber that is a part of microfluidic device and thatcontains the said particles in suspension, comprises the steps:

-   -   (a) providing an electromagnetic means to generate magnetic        field sequences having polarity and intensity that vary in time        and a magnetic field gradient that covers the whole space of the        said reaction chamber;    -   (b) applying a first magnetic field sequence to separate or        confine the particles so the particles occupy a sub-volume in        the volume of the reaction chamber;    -   (c) injecting a defined volume el the said reagent in the        reaction chamber;    -   (d) applying a second magnetic field sequence to lead the        particles to be homogenously distributed and dynamically moving        as a fog of particles occupying a substantial portion of the        whole reaction chamber volume;    -   (e) leaving the fog of particles in the homogenous state for a        defined period of time to allow a reaction to take place between        the particle surfaces and the reagent injected in the said        reaction chamber; and    -   (f) in the case of a large reagent volume, repeating the steps        (b)-(e) until a given reagent volume has passed through the        reaction chamber.

Another key aspect of the present invention concerns the magnetic polesactuation mechanism which consists of:

-   -   (1) applying from the electromagnetic poles magnetic field        sequences having polarity and intensity that vary in time; said        varying magnetic field sequences being effective to break or        inhibit particle claim aggregates and to maintain the particles        in suspension as a fog of particles in relative dynamic motion;        and    -   (2) combining the magnetic fields from different magnetic poles        in a sequence to induce displacement of the fog of particles        across the reaction chamber whereby the fog of particles        occupies substantially the whole reaction chamber volume        quasi-instantaneously or over a period of time.

Accordingly, the present invention concerns a method of mixing magneticparticles in a rnicrofluidic environment with surrounding medium in areaction chamber that is a part a rnicrofluidic network, wherein atleast a couple of electromagnetic poles face each other across thereaction chamber, arranged to provide a magnetic field gradient over thewhole volume of the said reaction chamber. The key element of theinvention is related to the magnetic poles actuation mechanism which isbased on the application in each electromagnetic pole of magnetic fieldsequences having polarity and intensity that vary in time. It has beenfound that this mechanism of magnetic poles actuation leads tocontinuous time variations of the position (displacement) of themagnetic field gradient maxima across the reaction chamber volume,leading thereby the particles to be in perpetual relative translationaland rotational motion that can substantially cover the whole reactionchamber volume.

Additionally, the desired effect obtained by the actuation mechanismaccording to the invention is that during their motion the particles donot displace as a compact aggregate but they are rather moving in as afog of particles resulting in a strong enhancement of the contactbetween the particles surfaces and the surrounding liquid medium.

Additionally, the desired effect obtained by the actuation mechanismaccording to the invention is that the particles mixing will coversubstantially the whole reaction chamber volume and not be limited to anarrow segment as in the disclosed prior art concepts. This magneticparticles handling process advantageously assures therefore a homogenousmixing allowing much more liquid volume to be effectively contacted withthe particle surfaces.

Additionally, the desired effect obtained by the actuation mechanismaccording to the invention is the possibility of selecting the magneticfield sequence to not only homogenously mix the particles but alsoseparate or confine the particles so the particles occupy a sub-volumein the volume of the reaction chamber as the outer borders of thereaction chamber. For instance one can apply a first magnetic fieldsequence to homogenously displace and therefore mix the particles insubstantially the whole reaction chamber volume; and then apply a secondmagnetic field sequence that specifically selects the direction of themagnetic field gradient leading the particles to be drawn to asub-volume of the reaction chamber determined by the direction of theapplied magnetic field gradient. This flexibility in controlling theparticles is advantageously important as it allows to handle and controlthe particles state in correspondence with the assay process.

Another feature of the invention is that during their perpetual motion(movement) the size of the particles aggregates can be mainly controlledby the frequency of the magnetic field polarity oscillations while thehomogeneity of mixing is controlled by the magnetic field amplitude.Accordingly, the magnetic field (gradient) amplitude can be used as aswitching parameter between, for instance, a homogenous mixing stateover the reaction chamber volume and a separated state at the outerborder of the reaction chamber.

Additionally, a desired effect obtained by the device and the actuationmechanism according to the invention, is the extremely fast manipulationof the particles. For instance, starting from a configuration where theparticles are first separated to the outer border of the reactionchamber using a specific first actuation sequence, and a fraction oftime (a second or less) is sufficient to put the particles in ahomogenous mixing configuration using a second actuation sequence. Theparticles can afterwards again be drawn in a fraction of time to theouter border of the reaction chamber by applying the first actuationsequence. This rapid manipulation process can be even reached in acomplex high viscous medium like blood lysate.

According to the previously described aspects and effects of handlingmagnetic particles, the inventive method further includes the steps ofconducting an assay wherein: (1) the particles are separated or confinedin a sub-volume of the reaction chamber as the reaction chamber externalborders using a first magnetic poles actuation sequence; (2) followed bythe injection of a defined volume of an assaying reagent that preferablywill not exceed the reaction chamber volume; (3) as the liquid flow isstopped, the particles will be mixed to be substantially homogenouslydistributed over the reaction chamber volume using an appropriateactuation sequence; (4) after mixing for a defined time period to allowthe desired reaction between the particles surfaces and the injectedassay reagent to take place, the particles will be attracted again tothe reaction chamber walls and the new sample volume injected to thereaction and then mixed; and (5) this process will be repeated insequential way until a defined volume of the assay reagent is mixed withthe magnetic particles.

One; of the advantages of such “separation/injection/mixing” mode is theassay process, performed in “discontinues batches” of a volume equal tothe reaction chamber volume. The small scale of the reaction volumealong with the rapid and effective mixing and separating the magneticparticles according to the invention, allow for an efficient and rapidconduction of the assay batches and thereby the overall assay process.

To achieve the previously described method and the related effects, thereaction chamber is preferably a cavity that has an inlet port and anoutlet port and at least one segment with diverging/converging partsconnected respectively to inlet and outlet port for delivering liquidsinto and from the reaction chamber. Moreover, the said reaction chambercan be inserted in the air gap of electromagnetic poles that aregeometrically arranged in a way to be co-diverging/co-converging withdiverging/converging parts of the reaction chamber. The saidelectromagnets are arranged to provide a magnetic field gradient overthe whole volume of the reaction chamber.

To achieve the previously described method and the related effects,preferably the magnetic poles are each electromagnetically actuatableindependently from each other and wherein each couple of magnetic polesform a closed magnetic circuit with a magnetic gap in which the saidreaction chamber is located.

To reach the desired effects, preferably the magnetic particles areinitially unmagnetized magnetic particles that develop a specificferromagnetic hysteresis response to an external magnetic field. Morespecifically, the particles have a coercive field between 200 to 1000Oe.

To reach the desired effects, the time varied magnetic field sequencescan preferably have a substantially rectangular, sinusoidal, saw-tooth,asymmetrical triangular, or symmetric triangular form, or anycombination of such forms, with a frequency that is preferably greaterthan 1 cycle per second and a maximum amplitude that is lower than thecoercive field of the magnetic particles in use.

According to one embodiment of the present invention and based on all ofthe previous aspects of mixing magnetic particles, a method to extracttarget molecule(s) that enter in the composition of intracellularcomplexes in a sample volume comprises:

-   -   (a) providing a reaction chamber that is part of a rnicrofluidic        device and that contains a first magnetic particles type in        suspension; wherein the said first type particles have a surface        coating designed to selectively bind with the said target        molecules;    -   (b) providing an electromagnetic means to generate magnetic        field sequences having polarity and intensity that vary in time        and a magnetic field gradient that covers the whole space of the        said reaction chamber;    -   (c) applying a first magnetic field sequence to separate or to        confine the said first particles type so the particles occupy a        subvolume in the volume of the reaction chamber;    -   (d) injecting in the reaction chamber a defined volume of the        said sample, wherein the cells are previously labelled with a        second type of magnetic particles;    -   (e) applying a second magnetic field sequence to lead the said        first particles type to be homogenously distributed and to        dynamically moving as a fog of first particles that occupies a        substantial portion of the whole reaction chamber volume.    -   (f) leaving the fog of first particles type in the homogenous        state for a defined period of time to allow strong contact        between the first particle type surfaces and the said        magnetically labelled cells injected in the reaction chamber,        thereby forming by means of dipolar interaction a complex        composed from the first particles type and magnetically labelled        cells;    -   (g) applying a further magnetic field sequence to separate or to        confine the said complex in a specific area of the reaction        chamber;    -   (h) in the case of a large sample volume, repeating the steps        (c)-(g) until a given sample sub-volume is passed through the        reaction chamber; and    -   (i) lysing the so separated cells to release the said target        molecules in the reaction chamber in conditions that allows        specific capturing of the target molecules on the first        particles types surfaces.

Accordingly, the disclosed method of extracting a target molecule(s)such as nucleic acids or proteins that enter in the composition of cellssuch as bacteria or cancer cells in a sample volume, allows to combinecomplex analytical procedures as cells selection and separation followedby the cells lysis and target molecules purification, in one reactionchamber.

In practice, this method consists first of the cell selection using afirst type of magnetic particles having a surface coating designed toallow affinity recognition of the said cells. The so-labelled cells arethen magnetically separated in a microfluidic chamber where secondmagnetic particles type are manipulated according the inventive methodof mixing magnetic particles. The separation process is performedfollowing the “separation/injection/mixing” mode, wherein the separationprocess is allowed by the efficient mixing to strongly contact themagnetically labeled cells and the second magnetic particles type. Oneaspect, of the cell separation method, indeed, is the formation of acomplex composed from the first particles type and magnetically labelledcells by means of dipolar interaction during the mixing process of thefirst particle type. As this complex is formed, the separation of themagnetically labelled cells from the supernatant can be performed duringthe separation of the first magnetic particles type.

Regarding a final objective of the invention, the particles in use havea surface coating designed to allow affinity recognition with at leastone target molecule or reaction with the surrounding liquid mediumwithin the reaction chamber. The said target molecules or reagents arecarried by a flow to the reaction chamber. When combined together, allaspects of the current invention allow the processing with enhancedperformance of complex bio-chemical, synthesis and analysis proceduresusing magnetic particles as a solid support. Typical examples butwithout limitation of such procedures are enzymes-linked assay, proteinsand nucleic acids extractions, or detection methods based on enzymaticsignal amplification methodologies such as chemioluminescence, NASBA,TMA or PCR.

BRIEF DESCRIPTION OF THE DRAWINGS

The objects and features of the present invention are set forth withparticularity in the appended claims. The present invention, both as toits organization and manner of operation, together with further objectsand advantages, may best be understood by reference to the followingdescription, taken in connection with the accompanying drawings, wherein

FIGS. 1 (a) and (b) are schematic representations of the concept offlow-through magnetic particle handling approaches as described in theprior art.

FIG. 2 (a) shows a schematic view of one preferred embodiment of theinvention which includes one couple of “diverging” magnetic poles facingeach other across a gap and a reaction chamber (channel) placed in thisgap. FIG. 2 (b) is a cross-sectional view of FIG. 2 (a), showing inparticular the electromagnetic circuit that provides the magnetic fieldin the reaction chamber.

FIG. 3 (a) shows a schematic representation of another preferredembodiment of the invention which includes in particular one couple of“diverging” magnetic poles and a reaction chamber that has a divergingcavity arranged co-divergently with the gap geometry. FIG. 3 (B) showsthe magnetic field variation profile along the axis of the said magneticpoles.

FIGS. 4 (a) and (b) show the flow velocity profile and variation inducedby the diverging reaction chamber geometry.

FIGS. 5 (a), (b), and (c) are schematic representations of magneticparticle handling and mixing according to a preferred embodiment of theinvention which includes in particular a change in the direction of thepolarity of the magnetic poles, whose induced magnetic field has theeffect of axially moving the particles.

FIG. 6 shows a schematic view of yet another preferred embodiment of theinvention which includes a quadrupole configuration of magnetic poles,co-diverging/co-converging with the reaction chamber cavity.

FIGS. 7 (a) to (d) schematically represent, for the preferred embodimentof FIG. 6, the relative position and motion of the particles across thereaction chamber volume as a consequence of the actuation sequences ofthe electromagnetic poles using a magnetic field having a polarity andamplitude that vary with time.

FIG. 8 schematically represents the desired effect obtained with thequadrupole embodiment according to the invention where the particlesmixing and movement homogenously cover the whole reaction chambervolume.

FIGS. 9 (a) and (b) show a perspective view of the electromagneticcircuit according to a preferred embodiment of the invention.

FIG. 10 show a layout of a microfluidic chip according to a preferredembodiment of the invention.

FIGS. 11 (a) to (c) schematically represent a process of using theinventive mixing method and device for performing an assay in generaland an immunoassay in particular.

FIG. 12 schematically illustrates another embodiment of handling andmixing magnetic particles with the surrounding medium in a“pulsed-injection/mixing” mode.

FIG. 13 illustrates different behavior of the magnetic particles under arotating magnetic field.

FIG. 14 schematically represents another configuration of the magneticpoles and their operation to obtain the desired effects according to theinvention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The main attainable effect of the present invention is an effectivecontrol of the magnetic particles that allows an enhanced and homogenousmixing with the surrounding medium. In particular, the mixing of themagnetic particles is realized in a reaction chamber that is a part of amicrofluidic network and where the particles are handled using externalmagnetic poles with specific configurations and geometries. Accordingly,the different reagents are introduced to the reaction chamber usingliquid flows and the magnetic poles are specifically actuated to controlthe magnetic particles in use inside the reaction chamber.

Another main attainable objective of the present invention is a methodof biological liquid samples and reagents processing in a microfluidicenvironment in combination with their mixing with magnetic particles.

In general, the microfluidic environment of the invention concernsdevices typically designed on a scale suitable to analyze micro-volumespreferably in the range 0.1.mu.1 to 500 μl. However, in one of majorapplication of the invention large samples are used to concentratespecific biomolecules in the device to a small volume for subsequentanalysis. The microscale flow channels and wells have preferred depthsand widths on the order of 0.05-1 mm. The “reaction chamber” that ispart of a microfluidic network as used herein refers to chambers with acavity that have a volume in the range of 0.1 ml to 500 ml andpreferably in the range of 10 ml to 100 ml. However, for manyapplications, larger “mesoscale” dimensions on the scale of millimetresmay be used. Similarly, chambers in the substrates often will havelarger dimensions than the microchannels, on the scale of 1-10 mm widthand 1-5 mm depth.

In general, the microfluidic environment of the invention concernsdevices typically designed on a scale suitable to analyze micro-volumespreferably in the range 0.1 ml to 500 μl. However, in one of majorapplication of the invention large samples are used to concentratespecific biomolecules in the device to a small volume for subsequentanalysis. The microscale flow channels and wells have preferred depthsand widths on the order of 0.05-1 mm. The “reaction chamber” that ispart of a microfluidic network as used herein refers to chambers with acavity that have a volume in the range of 0.1 μl to 500 μl andpreferably in the range 10 μl to 100 μl. However, for many applications,larger “mesoscale” dimensions on the scale of millimeters may be used.Similarly, chambers in the substrates often will have larger dimensionsthan the microchannels, on the scale of 1-10 mm width and 1-5 mm depth.

To overcome the limitations of the prior art, a new magnetic device andmagnetic pole geometry is disclosed. Accordingly, as shown in FIG. 2, adevice for manipulating and mixing magnetic particles in a surroundingliquid medium, comprises: (i) at least one couple of magnetic poles(1)-(1′) facing each other across a gap, the facing poles diverging froma narrow end of the gap to a large end of the gap. The poles form partof an electromagnetic circuit and are arranged to provide a magneticfield gradient (5) in the gap region. In this gap region is placed atubular reaction chamber (2) that is a part of a fluidic network and inwhich the magnetic particles in use will be manipulated. The magneticcircuit is composed of a magnetic core (6) and coils (7) that whensupplied with an electric current produce a magnetic field in the gapregion through the magnetic poles (1), (1′). Moreover, each magneticpole (1) and (1′) is preferably electromagnetically actuatableindependently from each other using two independently actuatable coils(7).

The effect obtained by the described magnetic pole geometry is that themagnetic field gradient will not be limited to a narrow region but willcover the whole space region extending along the axial X direction inthe said poles air-gap.

To enhance the mixing effect, preferably the reaction chamber (2) placedin the air gap region has a cavity shape that varies in the samedirection as the geometry of the magnetic poles. As schematicallyrepresented in FIG. 3(a), rather than having a flow channel with auniform geometry (uniform cross-section) as in FIG. 2(a), the reactionchamber (2) has preferably a variable geometry that is substantiallyco-diverging in the diverging gap between the poles. With such variationof the reaction chamber geometry one will induce a transverse velocitygradient (8) leading to more effective flow mixing (see FIG. 4).

In operation, the space-varied magnetic field generated by the magneticpoles (1)-(1′) provides a magnetic field gradient and thus a magneticforce (5) along the X direction that will be used to retain the magneticparticles (9) during the flow of a fluid in the reaction chamber (2)(see FIG. 5). In order to be able to retain the particles (9) in thereaction chamber (2), the generated magnetic force (5) acting on theparticles must be greater than the flow drag force which tends to drivethe particles away. Moreover, since the magnetic force (5) and the flowdrag force decrease in the same way along the X direction, it will bepossible to control the generated magnetic force (5) so that it issubstantially equal to the flow drag force. When introduced in thereaction chamber (2) and subjected to a static magnetic field (5), themagnetic particles (9) tend to form magnetic chains along the magneticfield flow line. Due to the magnetic field gradient generated in thereaction chamber (2), the magnetic particle chains will coalesce to forma strongly aggregated chain-like structure. Preferably the amount ofmagnetic particles (9) used is such that the magnetic aggregatedstructure mostly located near the magnetic poles in the conical part ofthe reaction chamber (2), as shown in the left of FIG. 5 (a). A timevaried magnetic field as an alternating magnetic field is then appliedto break down the aggregated chain-like structures with the fluid flowthrough such magnetic particle structures at a predefined flow rate(i.e. slightly increased, as necessary). A low aggregated magneticstructure is obtained, as illustrated in the right of FIG. 5, andcontrolled by adjusting the magnetic field amplitude and frequency, themagnetic field gradient provided by the pole geometry, and the fluidflow rate in the reaction chamber (2).

The desired effect obtained by the so-described reactionchamber/magnetic pole geometries is that the magnetic field gradientvariation profile corresponds to the same variation profile as for theflow velocity gradient in the reaction chamber (as shown in FIG. 3(b)and FIG. 4(b)). Such “co-variation” of the flow velocity/magnetic fieldgradients (forces) allows to reach more homogeneity in the mixingconditions (and therefore more controlled and efficient mixing) of themagnetic particles with a liquid flow.

The geometrical parameter of the device of FIGS. 2 to 5 according theinvention, must be set in a way to reduce the magnetic losses and assurea maximum focus of the magnetic flux in the reaction chamber (2).Moreover, the adjustment of these dimensions must be performed in a waythat the generated magnetic field gradient covers the whole reactionchamber and minimizes the existence of zones inside the reaction chamberwith a vanishing magnetic field gradient. In this perspective, ratiobetween the depth (H) of the large end to the length (L) of thediverging part of the reaction chamber is between 0.1 to 10 andpreferably between 0.1 to 1. Typical values of the length (L) of thediverging parts are between 50 μM and 10 mm, preferably between 100 μmand 5 mm. The dimensions of the microfluidic channel connected to thenarrow end of the reaction chamber are in the range of 50 μm to 5 mm andpreferably between 100 μm and 1 mm.

Accordingly, a key aspect of the present invention concerns the magneticpoles actuation mechanism which is based on the application in eachelectromagnetic pole of magnetic field sequences having polarity andintensity that vary in time.

A typical example of this actuation aspect according to the invention isillustrated in FIG. 5. As schematically shown in FIG. 5(a), a “paralleloscillating” magnetic field (4)-(4′) is applied to the magnetic poles:

Poles 1 and 1′:B=Bo.sub.0 sin(2π,f ₁ t)  (1)

under such condition and due to the perpetual change in the fieldpolarity, the magnetic agglomeration (chains) will break down to smallerparticles chain-like structures with a size that decreases with thefield frequency (f₁). Ultimately, the particles will behave like a fogof particles in relative dynamic motion. Another important phenomenoncharacterizing the use of “oscillating” magnetic field, is thegeneration of negative dipolar interaction between the particles (due tothe fact that the particles will not rotate at the same rate) thatcontribute further in the particles agglomeration break-up. Forinstance, contrary to the case of a static field where the particleswill be mostly attracted as an aggregated mass toward the magnetic poles(as shown in FIG. 1(a)), in an oscillating magnetic field the particles(while rotating) tend to be homogenously distributed over the reactionchamber cross section (as shown in FIG. 5(a)). In other words, under anoscillating magnetic field the particles will tend to occupy a largerspace due the development of repulsive magnetic forces between theparticles.

In summary, the use of a magnetic field that has a polarity andamplitude that vary in time as a base actuation of the magnetic polesaccording to the invention allows for an effective breaking orinhibiting of particle aggregates and tends to maintain the particles insuspension as a fog of particles in relative dynamic motion.

However, as the manipulation of magnetic particles necessitates the useof magnetic (force) gradient (5), the particles will be attracted to thenarrow segment of the reaction chamber, which will confine and thereforetend to agglomerate the particles. This agglomeration can be reduced byreducing the applied field amplitude (B₀) and thereby the magnetic forcegradient. If in fact, one reduces the force by reducing applied fieldamplitude (B₀), one observes that the “rotating” particles structure (9)will expand radically along the X direction due to the repulsivemagnetic forces between the particles induced by their relativerotation.

To overcome further agglomeration induced by the magnetic fieldgradient, according to the invention as shown in FIG. 5(b), the polarityof the magnetic field polarity (4)-(4′) generated from each of magneticpoles (1) and (1′) is changed from parallel to opposite (anti-parallel):

Pole 1:B=B ₀ sin(2πf _(1t)+π)

Pole 1′:B=B ₀ sin(2πf _(1t))  (2)

to cause a change in the direction of the magnetic force (5), which willmove the particles (9) axially in the X direction, following thedirection of the magnetic force (5), from the narrow segment to thelarge segment of the reaction chamber.

Accordingly, continuous “switching” between the two actuation schemes ofthe magnetic poles defined by equations (1) and (2) leads to continuoustime variations of the position of the magnetic field gradient maximafrom the narrow to the large segments of the reaction chamber. Thesemagnetic field gradient maxima changes will in turn lead the particlesto be in perpetual axial movement between the narrow and the largesegments of the reaction chamber following the magnetic field gradient(5) variations.

Accordingly, the actuation mechanism according to the invention is basedon the finding that by appropriate choice of the switch frequency (f₂)between the actuation scheme of the magnetic poles defined by equation(1) and (2), one can reach a state where the particles willsubstantially homogenously cover the whole reaction volume, asschematically shown in FIG. 5(c).

The so described actuation mechanism, leads the particles to be inperpetual relative translational and rotational motion that cansubstantially cover the whole reaction chamber volume. Such particlesdynamics is the key factor in the disclosed particle mixing according tothe invention, as the mixing will cover substantially the whole reactionchamber volume and not be limited to a narrow segment as in thedisclosed prior art concepts. This magnetic particles handling processadvantageously assures therefore a homogenous mixing allowing much moreliquid volume to be effectively contacted with the particle surfaces.

Moreover, as when compared with the previous art magnetic particlesresuspension concept of E.P, Pat 0,504,192, the use of sequentialenergizing (actuation) of the electromagnets by “binary” (i.e., on andoff) or “analog” with the disclosed magnetic device of FIG. 2, leads theparticles to move very slowly while remaining mostly agglomerated.Moreover, the polarity alteration between the two states of FIGS. 5(a)and 5(b) will not substantially solve this issue as suggested by theU.S. Pat. No. 6,231,760. Such difficulties are a specificity of“microfluidics” where the relatively “small” working volume leads tostrong magnetic coupling between the adjacent magnets.

For solving this issue, the key finding of the present invention is toapply in each electromagnetic pole magnetic field sequences havingpolarity and intensity that vary in time, the role of which is toeffectively break or control the particle aggregates and to maintain theparticles in suspension as a fog of particles in relative dynamicmotion; and then combining the magnetic fields from different magneticpoles in a sequence to induce homogenous mixing of the particles oversubstantially the whole reaction chamber volume.

For clarity, and contrary to what one could as a first view expect, the“arrows” representing the magnetic poles polarity in FIGS. 5(a) and 5(b)(and in all other Figures), are not fixed. In practice, these polaritiesare continuously changing direction in time. The “fixed” narrowdirection, “instead”, represents the “relative” polarization of theelectromagnets during the particles manipulation.

In another embodiment according to the invention and as shown in FIG. 6,a device for manipulating and mixing magnetic particles is providedwhere the magnetic poles form a quadrupole comprising (i) a first coupleof magnetic poles (1)-(1′) facing each other forming a diverging gap;and (ii) a second couple of magnetic poles (10)-(10′) facing each otherand forming a diverging gap, with the large ends of the diverging gapsof the first and second couples of poles facing one another; and (iii) areaction chamber (2) that is a part of a fluidic network, having acavity with diverging parts of the reaction chamber that are arrangedco-divergently in the diverging gaps between the poles.

It is clear that the quadrupole configuration is a more sophisticatedversion of the previous embodiments allowing more enhanced effects. Morespecifically, the magnetic field gradient (5), rather than beingsubstantially axial (axial symmetry) as in the case of the previouslydescribed two poles configuration, has a substantially “sphericalsymmetry”. The possibility of having a “multi-directional” magneticfield gradient induced by more than a couple of magnetic poles, offersthe possibility to move the position of the magnetic field gradientmaxima following more “rich” configurations as shown in FIG. 7 (a)-(d).In particular, by proper and sequential actuation of the magnetic field(4) induced from each magnetic pole of the multi-poles (quadrupole)configuration one can to move the position of the magnetic fieldgradient maxima across the reaction chamber volume in way that thesequential position of these maxima covers the whole reaction chambervolume.

FIGS. 7(a)-(d) schematically represent the different magnetic polesactuation (4) and the corresponding magnetic particles configurations,which correspond indeed to the position of the magnetic field gradientmaxima. An effect obtained by such magnetic handling process is that bysequentially moving the particles, for instance, following theconfiguration of FIGS. 7(a)-(d), the particles movement will coversubstantially the whole volume of the reaction chamber as shown in FIG.8, thereby assuring a strong mixing with the surrounding liquid medium.

From what precedes, a first key element in the actuation mechanismaccording the invention is a “base” magnetic field actuation (4) of themagnetic poles which is a magnetic field with a polarity and amplitudethat vary with time. A typical example of this actuation field is anoscillating magnetic field as the one of equation (1). In general, suchbase magnetic actuation field has a substantially rectangular,sinusoidal, saw-tooth, asymmetrical triangular or symmetric triangularform or any combination of such forms.

From what precedes, a second key element in the actuation mechanismaccording to the invention is that the magnetic poles are actuatedfollowing a certain sequence to induce continuous time variations of theposition of the magnetic field gradient maxima across the whole reactionchamber volume, causing thereby the particles in use to be in relativedynamic motion covering the whole reaction chamber volume.

Accordingly, in the invention by “field sequences having polarity andintensity that vary in time” one means the composition of the “base”actuation field on each magnetic pole along with its sequentialvariation to induce the particles movement across the reaction chambervolume. in practice indeed, one can decompose the field sequencesactuating each magnetic pole in two main components: (1) a baseactuation field component that has a polarity and amplitude that varywith time and (2) a sequential variation of this base actuation field toinduce the particles displacement across the reaction chamber andthereby affecting particles mixing.

Accordingly, in practice the base actuation field component will havethe role of breaking the particles chains aggregates and thereby assurelarge surfaces of the particles to be in contact with the surroundingliquid medium while the sequential variation of this base actuationfield will induce continuous move of the particles “fog” over the wholereaction chamber assuring thereby an homogenous exposure of the“disaggregated” particles over substantially the whole volume of thereaction chamber.

Consequently, a desired effect obtained by the actuation mechanismaccording to the invention is that during their motion the particles donot displace as a compact aggregate but they are rather moving as a fogof particles resulting in a strong enhancement of the contact betweenthe of particles surfaces and the surrounding liquid medium.

In the previously described actuation mechanism, the time variation ofthe base actuation field as well as the sequence actuation of themagnetic poles is a non-periodic variation but it is preferably aperiodic variation. In the periodic case, the frequencies of the basefield (f₁) and the actuation sequence (f₂) can be in practice different(f₁*f₂). To reach the previously described particle mixing effects, theactuation sequence frequency (f₂) is lower than or at most equal to thebase field frequency (f₁). In general, to reach the previously-describedparticle mixing effects the time field variation of the base field (i.e.the time variations of the amplitude and the polarity of in eachmagnetic pole) is preferably higher or at least equal to the sequentialtime actuation of the magnetic poles.

The time variations of the magnetic field in accordance with theinvention, defined by the frequencies f₁ and f₂, is in the order of 0.1Hz to 1000 Hz and preferably between 1 Hz and 500 Hz, or other timescales characterizing non-periodic variations.

An advantageous effect obtained by the actuation mechanism according tothe invention is that particles will exhibit a dynamics movement thatsubstantially covers the whole reaction volume over a certain period oftime. According to the invention indeed, the particles will homogenouslycover at least 60% of the reaction chamber volume and preferably between80% and 99% of the reaction chamber volume. This homogenous coveragewill be achieved in period of time that is determined by the sequenceactuation time (or frequency) of the magnetic poles. In practice, thehomogenous mixing is achieved in a period of time between 10 s and 10 nsand preferable is and 10 ms. In preferred embodiments and depending onthe actuation field parameters the homogeneity of mixing will cover 99%of the reaction chamber over time.

To reach the desired effects, the magnetic particles in use arepreferably initially unmagnetized magnetic particles that develop aspecific ferromagnetic hysteresis response to an external magneticfield. More specifically, the particles have a coercive field between200 to 1000 Oe. Contrary to what is reported in the previous art wherethe particles in use are preferably “superparamagnetic”, it has beenfound that the fact that the particles exhibit a specific(ferromagnetic) hysteresis response is a key condition to achieve themixing effects according to the invention. In fact, as described before,the particles actuation mechanism consists in the use of a preferably ahigh frequency “oscillating” field as “base” actuation magnetic fieldcomponent on each magnetic pole to control and break down the particlesaggregates. At such high variation frequencies (f₁>1 Hz), the fact thatthe particles have hysteresis response allow them to follow such “rapid”field variations by physically rotating with the field oscillations.This particles rotation in a high frequency oscillating magnetic field(field having polarity and intensity that vary in time) is at the originof the particles desegregation process.

Moreover, to reach the desired effects, it has been found thatpreferably the particles in use are manipulated with an “oscillating”(field having polarity and intensity that vary in time) magnetic fieldwith an amplitude (maximum field strength) that is lower then thecoercive field of the particles in use.

Accordingly, the particles in use preferably are synthesized withproperties following the process disclosed in the patent applicationWO2006/056579, herein incorporated entirely as a reference.

In general the invention provides a method of integrating all of thepreviously described magnetic particles handling and mixing inmicrofluidic environment concepts, The method consists in the use of areaction chamber that is a part a microfluidic network, wherein: atleast one couple of electromagnetic poles face each other across thereaction chamber, the method comprising: (a) applying magnetic fieldsequences having polarity and intensity that vary in time from each ofthe electromagnetic poles, (b) combining the magnetic field from eachmagnetic pole to induce continuous time variations of the position ofthe magnetic field gradient maxima across the whole reaction chambervolume; and (c) causing the particles to be in relative translationaland rotational motion covering the whole reaction chamber volume.

To obtain the desired effect, the magnetic poles are preferablymagnetically coupled one to each other by a “closed” magnetic circuit. Atypical example of such magnetic circuit is illustrated in theperspectives views of FIG. 9. Indeed as shown in FIG. 10(a), for thequadrupole configuration of FIG. 6, each magnetic pole (1)-(1′),(10)-(10′) is connected to an electromagnet formed by a magnetic core(6) with a coil (7). Moreover, each magnetic core (6) is in contact witha “base” magnetic core part (6′) in form of an “8”. The “8” shape of thebase magnetic core (6′) assures that each magnetic pole pairconfiguration forms a closed magnetic circuit assuring thereby astronger circulation of the magnetic flux during the actuation processlike the one described by equation (1). Moreover, the fact that eachpair of magnetic poles form a “closed” magnetic circuit is essential tostrongly focus (concentrate) the magnetic flux and magnetic fluxgradient in the reaction chamber. Moreover, this condition isparticularly preferable to assure the mixing process and effects, inaccordance with invention, as previously described.

FIG. 9(b) shows a more sophisticated form of the quadrupoleconfiguration and magnetic circuit of FIG. 9(a), with an array ofquadrupole configurations to assure parallel actuation and handlingmagnetic particles according to the invention in four different adjacentreaction chambers. The same design and construction of a quadrupolearray can be extended for handling magnetic particles in a larger numberof reaction chambers.

Another aspect of the invention is related to a microfluidic chip thatintegrates the different geometrical aspects of magnetic particlesmanipulation and mixing described above. Accordingly, a microfluidicchip comprises: (a) reaction chamber (2) that is a part of a fluidicnetwork, containing the particles in use in suspension and having atleast one cavity with diverging/converging parts, (b) inlet (12) andoutlet (13) channels, for delivering liquids into and from the reactionchamber and connected respectively to the narrow segments of thediverging/converging parts, (c) an entries structure (14) placed on bothsides of the reaction chamber (2) to receive magnetic poles that arepart of an external magnetic circuit and wherein the magnetic poles aregeometrically arranged in a way to be co-diverging/co-converging withdiverging-converging parts of the reaction chamber.

In addition to the reaction chamber, the microfluidic chip of theinvention is configured to include one or more of a variety ofcomponents that will be present on any given device depending on itsuse. These components include, but are not limited to, sample inletports; sample introduction or collection modules; cell handling modules(for example, for cell lysis (including the microwave lysis of cells asdescribed herein), cell removal, cell concentration, cell separation orcapture, cell growth, etc.; separation modules, for example, forelectrophoresis, gel filtration, ion exchange/affinity chromatography(capture and release) etc.; reaction modules for chemical or biologicalreactions or alteration of the sample, including amplification of thetarget analyte (for example, when the target analyte is nucleic acid,amplification techniques are useful.

All the previously described embodiments and aspects of the presentinvention have as a main objective to enhance the reaction rate betweenany target substances within a liquid medium and the particle surfacessuspended in the said liquid. An effective mixing, will indeed have astrong impact on the performance of any biochemical process such as theextraction or (and) detection of biomolecules for example (but notlimited to) nucleic acids and proteins. Moreover, one key element of thedisclosed magnetic particles handling concept is that the particlesmanipulation procedure can be readapted or adjusted in correspondencewith the biochemical process in consideration.

Usually the surface of the magnetic particle is biochemicallyfunctionalized by specific ligands for the probing and manipulating ofbiomolecules and chemical substances using well-known techniques. Forthis, the magnetic particle surface comprises for example a functionalgroup or a ligand that is capable of binding to a target molecule or toclass of target molecules. Potential functional groups comprise but arenot limited to carboxylic acids, hydroxamic acids, non-adhesivecompounds, amines, isocyanates, and cyanides. Potential ligands comprisebut are not limited to proteins, DNA, RNA, enzymes, hydrophobicmaterials, hydrophilic material, and antibodies. More generally,examples of ligands suitable for use in the present invention include,but are not limited to, molecules and macromolecules such as proteinsand fragments of proteins, peptides and polypeptides, antibodies,receptors, aptamers, enzymes, substrates, substrate analogs, ribozymes,structural proteins, nucleic acids such as DNA and RNA and DNA/RNAhybrids, saccharides, lipids, various hydrophobic or hydrophillicsubstances, lipophilic materials, chemoattractants, enzymes, hormones,fibronectin, and the like. Such molecules and macromolecules may benaturally occurring or synthetic. The term ligand may also includelarger entities such as cells, tissues, entire microorganisms, viruses,etc.

Using the so functionalized particles, the mixing and separation processof the present invention has particular utility in various laboratoryand clinical procedures involving biospecific affinity binding reactionsfor separations. Such biospecific affinity binding reactions may beemployed for the determination or isolation of a wide range of targetsubstances in biological samples. Examples of target substances arecells, cell components, cell subpopulations (both eukaryotic andprokaryotic), bacteria, viruses, parasites, antigens, specificantibodies, nucleic acid sequences and the like.

Moreover, the mixing and separation process of the present inventionhave particular use in detection procedures including, but not limitedto polymerase chain reaction (PCR), real-time PCR, ligase chain reaction(LCR), strand displacement amplification (SDA), and nucleic acidsequence based amplification (NASBA).

An example of use of the disclosed magnetic particles handling andmixing devices/method is illustrated in FIG. 11. This Figure illustratesthe different steps of a sandwich immunoassay where: (a) in a first step(FIG. 11(a)) the particles coated with specific capturing probes will bemixed to homogenously cover the reaction chamber as previously describedin FIGS. 7 and 8. In this step the sample containing the targetbiomolecules is pushed with a liquid flow through the reaction chamber,For that purpose reaction chamber/magnetic pole “co-variation”geometries will assure (as shown in FIG. 3 and FIG. 4) the homogeneityof the mixing conditions of the magnetic particles with the liquid flow.All these conditions when tidily adjusted allow a strong capturingefficiency of the targets on the particles surfaces. (b) After a washingstep, as described in FIG. 11(b), a defined volume (substantially equalto the volume of the reaction chamber) of a reagent containing detectionprobes is injected in the reaction chamber. In this case the particlescan be again homogenously mixed with the surrounding medium allowingefficient capturing of the detection probe on the particle surfaces. (c)After a washing step, as described in FIG. 11(c) a defined volume(substantially equal to the volume of the reaction chamber) of a reagentdetection substrate is injected in the reaction chamber. In this casethe particles can be homogenously contacted and mixed with thesurrounding medium allowing efficient interaction between the substrateand the detection probes on the particle surfaces. Contrary to classicalimmunoassay tests where the detection signal is mainly induced bydiffusion, our mixing process allows a strong interaction between thedetection substrate and the particles surfaces covering the wholereaction chamber volume. A large enhancement of the detection signal canbe therefore generated in this way allowing the detection of low targetmolecules concentration within the starting sample (as blood or plasma).As shown in FIG. 11 (c), during the detection the particles can be drawn(separated) to the reaction chamber borders following the sequence ofFIG. 7(b).

In a different embodiment of the use of magnetic particles handling andmixing according to the invention, rather than having a flow-through forcapturing targets from a large sample volume (as for instance describedin the first step of the previous example 11(a)), a target concentrationcan be achieved in a more controlled way under a static (no-flow)condition. This embodiment, schematically illustrated in FIG. 12, isbased on the use of the concept of “pulsed injection” (instead ofcontinuous flow) of the sample in the reaction chamber followed by ahomogenous mixing of the particles. More specifically, in a first step(FIG. 12, top) the particles are attracted to the reaction chamber walls(using the actuation sequence of FIG. 7(b)) and retained while a definedvolume of the sample, that will not exceed the reaction chamber volume,is injected. In a second step (FIG. 12 bottom), the particles coatedwith specific capturing probe will be mixed to homogenously cover thereaction chamber as previously described in FIG. 8, but without anyflow. After mixing of a defined time period, the particles will beattracted again to the reaction chamber walls and the new sample volumeinjected to the reaction and then mixed. This process will be repeatedin sequential way until the full sample volume is mixed with themagnetic particles.

One of the advantages of such “pulsed-injection/mixing” mode is that onewill avoid to deal with the constraints of handling magnetic particlesin a flow-through condition which is actually very difficult to setup.Moreover, contrary to the flow-through case where the contact time isstill relatively extremely short, the mixing time can be more easilycontrolled in a pulsed mode and adapted in correspondence with thetarget molecules and the final use.

Additionally, we have experimentally observed that it is difficult toretain the particles in the reaction under flow-through conditions whenthe particles are manipulated in a homogenous fog at high fieldvariation frequencies where the particles are desagglomerated andtherefore mixing conditions are more favorable. In such more favorablemixing conditions, the particles losses are very important and thereforethe “pulsed-injection/mixing” mode is practically more appropriate.

With a view to minimize and even completely avoid particle losses duringthe assay process, the particles are separated in a portion of thereaction chamber where the magnetic field gradient is higher, to retainthe particles in the reaction chamber during the reagents injectionstep.

One favourable separation position of the particles is the outer borders(as in FIG. 12, top). In this position and after applying a time variedmagnetic field sequence”, necessary to effectively separate theparticles, a static magnetic field can be applied to agglomerate theparticles and thereby strongly fix them at the reaction chamber borders.

Another favourable separation position of the particles is the narrowcorner of the reaction chamber facing the injected flow channel aspreviously described in FIGS. 2 to 5. At this position indeed, theparticles are strongly retained under a high flow rate while at the sametime assuring a strong mixing with the fluid flow, which gives a clearadvantage to this position when for instance compared to the first one.

From what precedes, the “separation/injection/mixing” mode can beconsidered as sequential (discontinuous) micro-reaction process, wherethe reaction can be conducted in “small” reaction volume in the presenceof large particle “fog” surfaces provided by the disclosed mixingmethod. When taking in consideration the rapid switching (in a fractionof a second of time) between the full mixing and separation states ofthe particles, each batch or sequence can take typically between 1 to 5seconds. This means that for instance for a volume of the reactionchamber in the range of 10 μl to 100 μl, an effective high “flow-rate”of up 3 ml/min can be achieved.

With the rapid processing of the “separation/injection/mixing” mode,this method provides also enhanced assay performance as the reactiontakes places in a small volume with enhanced particles mixing, asdescribed herein.

In practice, the “separation/injection/mixing” mode can be principallyused advantageously to concentrate a target molecule from a large volume(up to 10 ml or even more), but it can be also used for potentially anyreagent and assay process such as but not limited to; washing,biomolecules labeling, detection of a target and/or elution of thetarget from the particle surfaces.

In a different embodiment, the magnetic particles handling and mixingaccording to the invention is used to concentrate specific cells andthen extract molecule(s) that enter in the composition of that cells ina sample volume consists.

The cell concentration process consists in: (1) providing in thereaction chamber a first type particle have a surface coating designedto selectively bind with the said target molecules; (2) These particleswill be mixed and separated following the previously described processand method using magnetic field sequences having polarity and intensitythat vary in time; (3) in a first step the particles are separated orconfined in a sub-volume in the volume of the reaction chamber as forinstance schematically represented in FIG. 12, top; (4) as separated, adefined volume of the assayed sample contains the cells that contain thetargets as part of its composition. At this stage and according to theinvention the “target” cells are previously labelled with a second typeof magnetic particles; (5) when introduced in the reaction chamber, themagnetically labelled cells are separated from the crude sample bymixing them with the first particles types. The mixing process is asdescribed before, and consists in the application of an appropriatemagnetic field sequence to lead the said first particles type to behomogenously distributed and dynamically moving as a fog of firstparticles that occupies a substantial portion of the whole reactionchamber volume (as shown in FIG. 12 bottom). By mixing the firstmagnetic type with the injected labelled cells the objective is topromote the contact between the two magnetic entities and thereby formby means of dipolar interaction a complex composed from the firstparticles type and magnetically labelled cells. As the complex betweenthe first particles type and magnetically labelled cells, the separationof the magnetically labelled cells will be “synthetically” assured; and(6) the “pulse-injection” can be repeated until a given samplesub-volume is passed through the reaction chamber and therefore thetarget cells separated.

As the so described cell concentration process: consists on the use ofthe mixing and magnetic handling process to separate magneticallylabelled using “magnetic” interaction between the cells and the mixedparticles. Usually indeed the magnetic cell separation necessitate theuse of high magnetic gradient separator with langue flow channels ormagnetic columns. As proposed herein, the cell separation method,indeed, is based on the formation of a complex composed from the firstparticles type and magnetically labelled cells by means of dipolarinteraction during the mixing process of the first particle type,facilitating thereby the separation of the magnetically labelled cellsfrom the supernatant during the separation of the first magneticparticles type. When compared with the previous art, this separation andconcentration process is performed in a small volume of the reactionchamber (between 10-100 μl), using relatively very low magnetic fieldforces and in a fraction of time.

When the cells are concentrated in the reaction chamber, in anadditional step, the cells are lysed to release its contents andparticularly the desired target molecule(s) to be separated andpurified. To do so, the lysis step is preferably performed in “buffer”conditions that allow specific capturing of the said target molecules onthe first particles types surfaces in the reaction chamber. This targetscapturing step is “naturally” performed by homogenously mixing theparticles over the reaction chamber volume.

In practice, this method consists first of the mil selection using afirst type of magnetic particles having a surface coating designed toallow affinity recognition of the said cells. The so-labelled cells arethen magnetically separated in a microfluidic chamber where secondmagnetic particles type are manipulated according the inventive methodof mixing magnetic particles. The separation process is performedfollowing the “separation/injection/mixing” mode, wherein the separationprocess is allowed by the efficient mixing to strongly contact themagnetically labeled cells and the second magnetic particles type. Asthe cells are concentrated in the reaction chamber, in a second step,the cells are lysed to release a least one target molecule entering inits composition in conditions that allow specific capturing of the saidtarget molecules on the first particles types surfaces in the reactionchamber.

In practice, this method consists first of the cell selection using asecond type of magnetic particles having a surface coating designed toallow affinity recognition of the said cells. The so-labelled cells arethen magnetically separated in a microfluidic chamber where firstmagnetic particles type are manipulated according the inventive methodof mixing magnetic particles. The separation process IS performedfollowing the “separation/injection/mixing” mode, wherein the separationprocess is allowed by the efficient mixing to strongly contact themagnetically labeled cells and the second magnetic particles type. Asthe cells are concentrated in the reaction chamber, in a second step,the cells are lysed to release a least one target molecule entering inits composition in conditions that allow specific capturing of the saidtarget molecules on the first particles types surfaces in the reactionchamber.

The sizes of the said first and second magnetic particles types arerespectively in the range of 0.1 μM to 500 um and 5 nm to 5 μm.Preferably with a size between 1 μm to 10 μm, the first particles typeare initially unmagnetized particles that develop a ferromagneticresponse with a hysteresis. Practically, the second particles type arepreferably selected with a size lower and at most equal to the size ofthe first particles type. Moreover, as the second particles type areused as a label for the target cells, their size is preferably taken inthe nanometre range, more specifically between 10 nm and 500 nm. Thesecond particles type can be, however, superparamagnetic but initiallyunmagnetized particles that develop a ferromagnetic response can be usedas well. In the latter case, can be advantageous to assure strongdipolar interaction during the cells separation and concentration step.

For the lysis step, one can use different means chemical lysis withguanidinium SCN, enzymatic lysis with proteinase K or lysostaphin,thermal lysis, use of ultrasound as well as electrical fields orelectromagnetic radiations, a strong pH gradient inducted by localizedelectrolysis and mechanical lysis. The lysis using physical means likeelectromagnetic radiations or an electric field as the lysis can beperformed in a favourable environment and conditions allowing thecapture of the target molecules on the particle surfaces. The saidconditions can be performed by adjusting for instance the pH or in thespecific buffer conditions such as in chaotropic or anti-chaotropicagents.

Regarding the final objective of the invention which is the integrationand automation of complex biochemical assays in an easy to use device,and to control the different steps and aspects described before, themicrofluidic system with the chip and magnetic device further comprisesa plurality of reagent sources fluidly connected to said reactionchamber; means for driving/controlling liquids like pumps and valves;and a computer controller for controlling reagent flow and applicationof the magnetic held sequences.

Even though all of the described methods and aspects are realizedthrough the previously described features as magnetic poles/reactionchamber, some of the effects disclosed here can be obtained by othermagnetic pole configurations. As described before, the main conditionsthat must be satisfied are (1) that the magnetic field gradientgenerated by the electromagnets must. cover the whole reaction chamberwith a defined localization of magnetic field (gradient) maxima when themagnetic poles are specifically actuated; (2) the magnetic fieldgradient must retain and confine the particles in the reaction chamberduring the manipulation process; and (3) the magnetic poles are in aconfiguration that tends to focus magnetic flux in the reaction chamberin a way that each pair of magnetic poles facing the gap can bemagnetically coupled through the said gap. To further enhance thiseffect, the poles are preferably part of a closed magnetic circuit.

Typical example of such configuration is the one illustrated by FIG. 14.The configuration comprises a quadrupole pole types configuration (19)that can be actuated as the same way as the converging/divergingmagnetic poles previously described. The quadrupole configuration (19)is preferably associated with two couples of magnetic poles placed atthe right (16) and the left (17) of the quadrupole configuration (19).The magnetic pole couples (16) and (17) are actuated (in oppositeconfiguration) in a way to generate a lateral magnetic field gradient(18)-(17) that tends to confine the particles in the quadrupole air gapregion. In the case of the converging/diverging magnetic polesconfiguration, the lateral confining magnetic field gradient is assuredby the magnetic poles geometry and therefore has the advantage to avoidthe need for additional magnetic poles. Further, to have the desiredmagnetic field flux focus in the reaction chamber and the magnetic fieldgradient covering the whole reaction chamber volume, the magnetic polesmust be in form of magnetic tips (of 1 to 5 mm) located close to eachother in a small gap of about 1 to 5 mm.

However, the magnetic poles configuration of FIG. 14, is also limited bythe fact that the volume of the region under which the particles can bemixed and handled is very small.

The following examples further describe in detail the manner and processof using the present invention. The examples are to be considered asillustrative but not as limiting of this invention. All manipulationsgiven in the examples are at ambient temperature unless otherwiseindicated.

Example 1 of Actuation Mechanism

The actuation sequences of FIG. 7, is an illustration on how themagnetic particles, while the magnetic poles are actuated using a timevaried (amplitude and polarity) magnetic field, is used as a basesequence, can be moved following the combination of the magnetic fieldfrom each magnetic pole. During this movement the particles willsubstantially cover the whole reaction chamber volume as a fog ofparticles thereby assuring mixing. Although represented as“discontinuous” sequences, the sequences of the particles as shown inFIG. 7 can be achieved with a “rotating magnetic field” following themagnetic poles actuation sequences:

Pole 1 and 10′:B=B ₀ sin(ft)

Pole 1′ and 10:B=B ₀(sin(ft+π/2)  (3)

In equation (3) the base sequence actuation in each magnetic pole is anoscillating field while the actuation process is assured by a phaseshift of π/2 between the diagonally coupled magnetic poles. In thisconfiguration the base and the sequence actuation fields have the samefrequency f.

In the actuation according to the sequences of equation (3), twoparticles regimes can be distinguished: a low frequency and highfrequency regime.

At low frequency typically for f<5 Hz, the particles will rotaterelatively “slowly” and the particles will move across the reactionvolume producing typically the sequences as schematically shown in FIG.7. The particularity of this regime is that the particles during theirmovement from one magnetic poles configuration to the other (see FIG.7), the particles will have enough time to “aggregate” in longermagnetic chains. For frequencies higher than 1 Hz, the particles willexhibit fast and strong dynamics that covers substantially the whole(>90%) reaction volume. However, at this regime of a rotating magneticfield the particles still “relatively” aggregated.

More disaggregated particle sate will be ultimately obtained at higherfrequencies of the rotating field f>5 Hz. At this regime instead theparticles behaviour is drastically different as the fast rotation of themagnetic particles will not give enough time for chain formation leadingthe particles chains to break down to smaller particles chain-likestructures with a size that decreases with the field frequency. As adifference with the low-frequency regime, the sequence of FIG. 7 (b)will not be observed as the particles will not have time to extend alongthe diagonal of the reaction chamber. What happens at high frequencyindeed is that the particles will be attracted and confined at thereaction chamber walls. FIG. 13 (a) shows a video of the particlesbehaviour at high frequencies.

To overcome this problem, a finding of this invention is to reduce theamplitude while increasing the frequency of the applied rotating fieldin combination with the use of ferromagnetic particles. The reduction ofthe magnetic field amplitude indeed allows to expand the particles moreover the reaction chamber volume due to reduction of the magneticgradient forces and the repulsive dipolar forces between the rotatingparticles. However, as the reduction of the magnetic forces will slowdown the particles movement, a higher frequency field is required tofurther propel the particles movement. At such high frequenciestypically between superior to 20 Hz and preferably in the range of 100Hz to 500 Hz, the use of ferromagnetic particles is key as the “magneticanisotropy” of these particles leads them to move and follow the fieldvariations. FIG. 13 (b), shows the homogenous coverage of the particlesin the reaction chamber obtained under a high rotating frequencies(around 300 Hz). During this mixing, the particles strongly move acrossthe reaction chamber allowing thereby strong and efficient mixing.

It is important to point out here that the frequencies values given inthis example are typical values just for indication, obtained withspecific particles used in experiments (MagNA Pure LC particles fromRoche Diagnostics). The use of other particles types will certainlyaffect the frequencies limits of different particles regimes andbehaviours as described before.

Example 2 of Actuation Mechanism

Equation (4) describes another actuation sequences to achieve mixingaccording to the invention.

Pole 1 and Pole 10′:B=B ₀ sin(f ₁ t)sin(f ₂ t)

Pole 1′ and 10:B=Bo sin(fit)sin(f ₁ t+π/2)  (4)

In this sequence indeed the first oscillation component (sin(fit)) isnothing more than the base actuation field at a frequency f₁ of themagnetic poles while the second term defines the actuation sequence thatmoves the “fog” of particles in rotation form with a frequency f₂. Thesequence of equation (4) allows in particular to solve the previouslyreported (in the Example 1) agglomeration of particles in a lowfrequency rotating field of equation (3). For instance by rotating theparticles as a frequency f₂=1 Hz, the particles chains will break downdue to the fast oscillation of the base field f₁>10 Hz.

Example 3 of Actuation Mechanism

Equation (5) describes another actuation sequence to achieve mixingaccording to the invention, where the frequency of the rotating magneticfield of equation (1) of equation is “modulated”

f=f ₀ +f ₁ sin(Ω·t)  (5)

The finding is that modulating the frequency between a low frequenciesregime and the high frequencies regime assures thereby efficient mixing.By appropriate choice of the modulating frequency (Ω), when can balancebetween the two regimes: homogenous mixing with agglomerations at lowerfrequencies and the “inhomogeneous” mixing with fog particles structureat higher frequencies. This way of “modulating” the frequency of therotating field is particularly important for highly viscous liquidswhere homogenous mixing is difficult to achieve by only increasing theoscillating frequency as described in Example 1.

It is obvious for skilled persons that the frequency modulation can bedone by other forms, as for instance a “square” signal where one switchbetween one high frequency value and a low frequency one. Each value canbe maintained for a certain time that depends essentially on the liquidviscosity, to assure an homogenous mixing.

It is worth to emphasize here again that the particles in use arepreferably ferromagnetic to allow the particles to move and rotate athigh frequency.

Example 4 of Actuation Mechanism

Although the previous examples are based on using “rotating magneticfield”, linear actuation sequence of particles fog can be also used tomix and reach an homogenous state. Typical example of that linearactuation mode can be achieved by first moving the particles to the outborder as shown in FIG. 7(b) using the actuation sequence:

Pole 1 and Pole 1′:B=B ₀ sin(ωt)

Pole 10 and 10′:B=B ₀ sin(ωt+π)  (6)

At this stage the particles can be moved to the left corner (narrowpart) of the reaction chamber by the sequence:

Pole 1 and Pole 1′:B=B ₀ sin(ωt)

Pole 10 and 10′:B=B ₀ sin(ωt+π/2)  (7)

By symmetry a displacement toward the right corner (narrow part) of thereaction chamber can be achieved by the sequence:

Pole 1 and Pole 1′:B=B ₀ sin(ωt+π/2)

Pole 10 and 10′:B=B ₀ sin(ωt)  (8)

A sequential shift between the previous three configuration followingthe sequences: (6)→(7)→(6)→(8) at a determined rate, one can achieve anhomogenous mixing over the time.

In practice, better mixing processes are achieved not through only arotating or a linear mode, but usually a mix of both modes is preferred.

Herein in these examples the choice of a “sinusoidal” field as baseactuation is only for it is practical analytical formulation with anequation. Within the invention scope, more complex actuation “basesequences” having polarity and intensity that vary in time will lead tothe same effects.

Example 5 of Use of the Mixing Concept and Device

In this example the disclosed magnetic particles device and method areused for DNA extraction from bacteria (E-coli) culture with an insertedplasmid. For the extraction, MagNA Pure LC kit from Roche Diagnostics(Switzerland) is used. A particularity of this kit is that the magneticparticles exhibit a ferromagnetic response with a coercive field ofaround 200 Oe.

For the sample preparation, 200 μl of the bacteria culture in PBS with aconcentration of around 2×10⁸ cells/ml are mixed with: (a) 400 μl oflysis binding buffer, (b) 100 μl of isopropanol, and (c) 100 μl ofProteinase-K. The total extraction volume is therefore 800 μl.

For the assay, a microfluidic chip with the layout of FIG. 11 is used.The reaction chamber in this chip has the following dimensions: H=0.25mm, L=0.5 mm and a depth of 1 mm. The total volume of the reactionchamber is therefore around 25 μl in this reaction chamber around 50 μlof the glass particles from the kit is separated and retained in thereaction chamber.

The samples and reagents processing through the chip is performedfollowing the previously described “pulse-injection” mode and where theparticles are homogenously mixed over the reaction chamber over a periodof 2 s followed by a separation and liquid injection of around 1 s.Around 3 seconds are necessary to process 25 pa of the sample volumewhich is equivalent to processing flow rate of 0.5 ml/min.

The washing step is performed using the three washing reagents of thekit with 3001 volume of each. The washing is performed by combining boththe flow-through mode and “pulse-injection” mode. Less than 2 minutesare necessary to perform all the necessary washing steps. For the DNAelution, a volume of the elution buffer from the kit substantially equalto the reaction chamber volume (−30 μl) and homogenously mixed foraround 3 minutes.

To determine the homogenous mixing benefits, the extraction performanceis compared with the standard manual extraction (as a reference) and thenon-homogenous mixing under a high frequency rotating magnetic field asdescribed in example 1 and shown in FIG. 13 (a). For the performance ofDNA extraction experiments we use the optical absorbance, with thefollowing results:

Total DNA amount Purity (μg) (OD 260/280) Manual extraction 6 1.7Homogenous mixing 5.5 1.9 Non-homogenous mixing 1.2 1.6

From these results one can see the strong impact of the proposedmagnetic particles mixing effect in enhancing the affinity bindingbetween the particles and the target molecule (DNA) in the sample. Infact, while the manual extraction takes around 20 minutes to beperformed around 8 minutes are necessary for full extraction using thedisclosed homogenous mixing method and device. Moreover, in the manualextraction around 100 μl of particles suspension is used while only 50μl is used in the microfluidic homogenous mixing. Taking inconsideration the relatively large amount of DNA that can be purified(up to 10 μg) in a small reaction chamber volume (25 μl) with thedisclosed homogenous mixing as disclosed herein, is clear expression ofthe large available surface of particles during the mixing demonstratingthe effective particles desegregation and mixing during the assay.Another demonstration of the particles homogenous mixing is the lowperformance obtained by non-homogenous mixing.

Example 5 of Use of the Mixing Concept and Device

In this example the disclosed magnetic particles device and method areused for DNA extraction from human whole blood. For the extraction,MagNA Pure LC kit II from Roche Diagnostics (Switzerland) is used withthe same process and protocol as Example 4.

The extraction results show a yield between 4-5 μg of DNA with an ODvalue between >1.7. This example, demonstrate the efficient DNAextraction of the disclosed mixing method from a complex sample likewhole blood.

Those skilled in the art will appreciate that various adaptations andmodifications of the just-described preferred embodiments can beconfigured without departing from the scope and spirit of the invention.Therefore, it is to be understood that, within the scope of the appendedclaims, the invention may be practiced otherwise than as specificallydescribed herein.

What is claimed is:
 1. A method of mixing magnetic particles with areagent in a reaction chamber that is pan of a microfluidic device andthat contains the particles in suspension, comprises the steps: a.providing an electromagnetic means to generate magnetic field sequenceshaving polarity and intensity that vary in time and a magnetic fieldgradient that covers the whole space of the reaction chamber; b.applying a first magnetic field sequence to separate or confine theparticles so the particles occupy a sub-volume in the volume of thereaction chamber; c. injecting a defined volume of the said reagent inthe reaction chamber; d. applying a second magnetic field sequence tocause the particles to be homogenously distributed and dynamicallymoving as a fog of particles occupying a majority of the whole reactionchamber volume; e. leaving the fog of particles in the homogenous statefor a defined period of time to allow a reaction to take place betweenthe particle surfaces and the reagent injected in the said reactionchamber, thereby forming a complex; and f. repeating the steps (b)-(e)until a given reagent volume has passed through the reaction chamber. 2.The method of according to claim 1, wherein the particles have a surfacecoating designed to selectively bind the particle with at least onetarget molecule in suspension within the reaction chamber.
 3. The methodaccording to claim 1, wherein the reaction chamber comprises amicrochannel.
 4. The method according to claim 1, wherein the reactionchamber comprises a cavity that has an inlet port and an outlet port andat least one segment with diverging/converging parts connectedrespectively to inlet and outlet ports for delivering liquids into andfrom the reaction chamber.
 5. The method according to claim 1, whereinthe magnetic means comprise at least two electromagnetic poles facingeach other across the reaction chamber and electromagneticallyactuatable independently from each other.
 6. The method of mixingmagnetic particles according to claim 5, wherein the said magnetic polesare geometrically arranged in a way to be co-diverging/co-convergingwith diverging/converging parts of the cavity.
 7. The method accordingto claim 1 wherein the time-varied magnetic sequence has a substantiallyrectangular, sinusoidal, saw-tooth, asymmetrical triangular, orsymmetric triangular form; or any combination of said forms.
 8. Themethod according to claim 7, wherein the oscillation frequency of themagnetic field is between 0.1 to 1000 cycles per second.
 9. The methodaccording to claim 1, wherein during the step (b) the particles areseparated or confined at the outer border of the reaction chamber. 10.The method according to claim 1, wherein during the step (c) theinjected reagent volume is equal to or lower than the reaction chambervolume.
 11. The method according to claim 1, which further comprises thesteps of: g. applying a further magnetic field sequence to separate orto confine the complex in a specific area of the reaction chamber; h.evacuating the reagent from the chamber; i. injecting another reagentinto chamber; and j. repeating the steps (b)-(e) of claim 1 until agiven reagent volume has passed through the reaction chamber.
 12. Amethod to extract target molecule(s) that enter in a composition ofintracellular complexes in a sample volume, said method comprises: a.providing a reaction chamber that is part of a microfluidic device andthat contains a first type of magnetic particles in suspension; whereinthe first type of particles have a surface coating designed toselectively bind with the target molecules; b. providing anelectromagnetic means to generate magnetic field sequences havingpolarity and intensity that vary in time and a magnetic field gradientthat covers the whole space of the reaction chamber; c. applying a firstmagnetic field sequence to separate or to confine the first type ofparticles so the particles occupy a sub-volume in the volume of thereaction chamber; d. injecting in the reaction chamber a defined volumeof the said sample, wherein the cells were previously bound to a secondtype of magnetic particles; e. applying a second magnetic field sequenceto cause the first type of particles to be homogenously distributed anddynamically moving as a fog of first particles over a substantialportion of the whole reaction chamber volume; f. leaving the fog of thefirst type of particles in the homogenous state for a defined period oftime to allow strong contact between the surfaces of the first type ofparticles and the said magnetically labelled cells injected in thereaction chamber, thereby forming by means of dipolar interaction acomplex composed of the first type of particles and the magneticallylabelled cells; g. applying a further magnetic field sequence toseparate or to confine the complex in a specific area of the reactionchamber; h. repeating the steps (c)-(g) until a given sample sub-volumeis passed through the reaction chamber; and i. lysing the complex torelease the target molecules in the reaction chamber to capture thetarget molecules on the first particles types surfaces.
 13. The methodaccording to claim 12, wherein the said target molecules are selectedfrom nucleic acids, proteins and peptides.
 14. The method according toclaim 12, wherein the said cells are selected from viruses bacterialcells, human cells, animal cells and plant cells.
 15. The methodaccording to claim 12, which further comprises the step of washing thetarget molecules captured on the particles from a residual liquidmedium.
 16. The method according to claim 12, which further comprisesthe step of eluting the captured molecules from the surfaces of theparticles.
 17. The method according to claim 12, which further comprisesthe step of detecting the target molecules.
 18. The method according toclaim 1, wherein the microfluidic device further comprises a pluralityof reagent sources fluidly connected to said reaction chamber; and acomputer controller for controlling reagent flow and application of themagnetic field sequences.
 19. The method according to claim 1, whichfurther comprises the steps of: (k) applying magnetic field sequenceshaving polarity and intensity that vary in time to disperse the magneticparticles; said varying magnetic field sequences being effective tobreak particle claim aggregates and inhibit the formation of particleclaim aggregates and to maintain the particles in suspension as a fog ofparticles in relative dynamic motion; and (l) combining differentmagnetic field sequences to induce displacement of the fog of particlesacross the reaction chamber whereby the fog of particles occupiessubstantially the whole reaction chamber volume.
 20. The methodaccording to claim 7, wherein during the step (b) the particle size andthe homogeneity of mixing the particles are controlled by varyingrespectively the frequency and the amplitude of the magnetic field. 21.The method according to claim 19, wherein the fog of particles occupiessubstantially the whole reaction chamber volume quasi-instantaneously.22. The method according to claim 19, wherein the tog of particlesoccupies substantially the whole reaction chamber volume over a periodof time.